Virus infection of susceptible cells requires interaction with cellular receptors. Many viruses undergo a biphasic binding to cells prior to entry. Typically an initial low affinity interaction can be detected when virus is added to cells at 4 degrees. A subsequent shift to 37 degrees is often accompanied by a strengthening of binding. Herpesviruses appear to be distinct from other virus families in that both initial attachment and stable adhesion to cells can occur at 4 degrees and does not require physiological temperature. Since membrane fusion requires 37 degrees in all enveloped viruses tested to date, HSV provides a novel, temperature-dependent system for dissecting the events that trigger the poorly understood process of virus-cell fusion. Current models of herpes simplex virus (HSV) entry attribute primary, low affinity attachment to cell surface glycosaminoglycans (GAGs). It has not been established which cellular molecule(s) mediates subsequent stable binding of HSV to cells. Determination of the cellular factors important for stable binding will facilitate understanding of molecular events leading up to HSV penetration of the host cell. Stable binding is defined experimentally as the ability of bound virus to resist removal by a selective, stringent wash (e.g. high salt, PBS-heparin). Recently, several molecules were identified which allow entry of HSV into nonpermissive cells and bind to viral glycoprotein D (gD) which is required for HSV infection. These herpesvirus entry mediatiors (Hve) are designated HveA (or HVEM), HveB, and HveC (or nectin1).To determine whether these molecules function to stabilize virus binding to cells, the effect of a heparin wash on purified, radiolabelled virions bound to cells was assessed. Binding of HSV to cells that expressed HveA or HveC was heparin-resistant (stable). Virus did not stably bind to HveB-expressing cells, which is consistent with the inability of HveB to mediate wild type HSV-1 entry. Stable binding was blocked by soluble coreceptors and by soluble recombinant gD, indicating that heparin-resistant attachment of HSV is specifically mediated by gD-binding coreceptors such as HveA and HveC . The results suggest that during virus entry, GAG-dependent binding of HSV to cells is stabilized by subsequent interaction with gD-binding coreceptors.